This invention relates to the cleavage of deoxyribonucleic acid (DNA) and, more specifically, to the cleavage of DNA by cyclic conjugated enediynes.
DNA is a very long, threadlike molecule which exists in the cells of all living organisms and which is intimately involved in the storage and transfer of genetic information. DNA is composed of discrete chemical units in a sequence unique to the particular organism from which it is derived.
DNA cleavage is currently a topic of considerable research investigation, due in a large part to the recognition that certain molecules interact and bind with sites on a DNA molecule on the basis of the site's specific chemical sequence. The sequence-specific cleavage of DNA is essential for many techniques in molecular biology, including gene isolation, DNA sequence determination, and recombinant DNA manipulation. Presently, such cleavage is performed largely with naturally-occurring restriction enzymes which bind and cleave DNA at particularly sequenced sites. However, because both the number and sequence specificities of such enzymes are limited, it is only possible to cleave DNA at a fixed number of recognition sites. It would be of great advantage, however, to be able to cleave DNA at any predetermined site; the design of sequence-specific DNA cleaving molecules that go beyond the specificities of natural enzymes might provide this capability. The ability to design molecules with predetermined specificities for selective cleavage would be of great importance for drug design, molecular biology, and materials chemistry.
The capability to selectively target a particularly sequenced site on DNA and modify it in some manner may provide a means of treatment for a disease or condition controlled by that site. For example, it has long been the desire of medicine to kill cancer cells in man. Conceivably, such cells might be killed if their DNA were properly cleaved. A number of molecules are known to facilitate DNA strand cleavage; however, many such compounds are believed to attack DNA in a non-selective fashion. Because of the toxic nature of such non-selective DNA-reactive compounds, medicinal therapies which employ them have generally been reserved for advanced forms of cancer and other life-threatening diseases. With the advent of molecules which can selectively bind and cleave cancer cell DNA on the basis of a particular chemical sequence, new methods for the design of safe, effective, and highly specific therapeutic anticancer agents might be developed. Much effort has therefore been directed toward the development of molecules that target and cleave chemically specific sites along a strand of DNA. Both naturally-occurring and synthetic compounds have demonstrated the ability to cleave DNA under certain conditions; however, the presence of other reactive species has in many cases been necessary to effect the desired cleavage. The recently reported calichemicin and esperimicin classes of antibiotics are novel naturally occurring compounds with unusually high potency against murine tumors. This potent antitumor activity has been attributed to the demonstrated ability of compounds of these classes to induce double-stranded DNA cleavage. However, the mode of action for compounds belonging to these classes is thought to depend upon the attack of some nucleophilic species. After recognition and interaction of a given molecule with DNA, the nucleophile is thought to trigger a sequence of intramolecular reactions centering on the bicyclic core structure characteristic of the calichemicin/esperimicin class. These reactions are believed to generate a highly reactive benzenoid diradical species through the cyclization of the conjugated enediyne moiety present within the bicyclic core. This species, in turn, is thought to react with and damage DNA's phosphate backbone.
The calichemicin/esperimicin class are characterized by large, complex molecular frameworks. A number of smaller molecules modeled after these antibiotics have been reported. Schreiber and Kiessling, J. Am. Chem. Soc., 110, 631 (1988), have described a procedure for the synthesis of bicyclic molecules comprising a conjugated enediyne moiety and a bridgehead double bond whose saturation is believed to play a central role in the mechanism for DNA cleavage. Magnus and Carter, J. Am. Chem. Soc., 110, 1625 (1988), have reported the synthesis of a cyclic cobalt complex which they hypothesize to generate--as a nonisolable intermediate--a bicyclic system similar to that of the calichemicin/esperimicin class, but differing in that its bridgehead position is already saturated. It is believed that this nonisolable intermediate generates benzenoid diradicals by way of a cyclization involving conjugated enediyne functionality.
Although the discussed calichemicin/esperimicin model systems have been constructed, no associated DNA-cleaving properties associated have been reported.
It has been widely hypothesized that the carbon-carbon double bond at the bridgehead of the discussed bicyclic system is a key element in the mode of action of the calichemicin/esperimicin class and a key determinant of any similar activity exhibited by systems modeled after them. This double bond locks the molecules into a certain geometry and prevents the ends of the diyne from approaching one another closely enough for cyclization and diradical generation to occur. Therefore, an intramolecular addition reaction which changes the double bond to a single bond--and thus allows molecular geometries suitable for cyclization--is thought to be a necessary first step in the DNA cleaving action of compounds incorporating such a bicyclic system into their molecular framework.